1. Field of the Invention
This invention relates to nonradioisotopically-labeled proteins and polypeptides useful as labeled conjugates in specific binding assays for determining such proteins and polypeptides, or specific binding partners thereof, in liquid media such as body fluids, particularly serum. In particular, the present invention relates to .beta.-galactosyl-umbelliferone-labeled proteins (e.g., immunoglobulins) and polypeptides useful in nonradioisotopic immunoassays.
2. Description of the Prior Art
Nonradioisotopic specific binding assays employing an enzyme-cleavable substrate label are described in German Offenlegungschriften Nos. 2,618,419 and 2,618,511, corresponding respectively to U.S. patent applications Ser. Nos. 667,892 and 667,996, both filed Mar. 18, 1976, and assigned to the present assignee. The assays avoid the use of radioisotopic labels and can be performed in homogeneous or heterogeneous formats. In the heterogeneous format, the bound- and free-species of the labeled conjugate are physically separated and the label measured in one of the separated species, whereas in the homogeneous format, the label expresses a different activity in the bound-species compared to the free-species, permitting performance of the assay without a separation step. In the aforementioned assays, the labeled conjugate serves as a substrate for a cleaving enzyme, with cleavage of the conjugate producing a distinguishable indicator product, usually a fluorescent product. The fluorescers umbelliferone or fluorescein are coupled to the ligand under assay through an ester bond which upon cleavage by an esterase releases the free fluorescent products, umbelliferone and fluorescein, respectively.
An improved substrate-labeled specific binding assay is described in pending U.S. patent application Ser. No. 886,094, filed Mar. 13, 1978, and assigned to the present assignee. The improvement comprises employing as the label component of the labeled conjugate, a residue of the formula: EQU G--D--R
wherein G is a glycone, D is a dye indicator moiety, and R is a linking group through which the dye indicator moiety is covalently bound to the binding component (usually the ligand under assay or a binding analog thereof) of the labeled conjugate. The cleavage enzyme employed is a glycosidase which cleaves the bond between the glycone and the dye indicator moiety, releasing a detectable, usually fluorescent, fragment comprising the dye indicator moiety coupled to said binding component (e.g., the ligand). Most preferably, the glycone is a .beta.-galactosyl group and the dye indicator moiety is umbelliferone.
Various .beta.-galactosyl-umbelliferone-labeled ligand conjugates are specifically described in the aforesaid Ser. No. 886,094 wherein the labeled ligand is a nonproteinaceous hapten of molecular weight less than 1000. It is highly desirable to prepare .beta.-galactosyl-umbelliferone-labeled conjugates for proteins and polypeptides of clinical significance so as to enable the homogeneous, nonradioisotopic specific binding assay determination of such proteins and polypeptides, and their binding partners. Preparation of such labeled protein and polypeptide conjugates is complicated by the complex structure and heterogeneity of proteins and polypeptides; the molecular size, fragility, and suceptibility to denaturation of such ligands; the need to maintain water solubility in the labeled conjugates; the need to maintain proper configuration in the conjugated protein or polypeptide; and the expected instability of chemically modified proteins and polypeptides over long storage periods.
The numerous conventional methods for modifying proteins and polypeptides and for coupling such ligands to solid supports and other materials are described in the following: for general reviews see Methods of Enzymology, vol. XLIV "Immobilized Enzymes," ed. Mosbach, Academic Press (New York 1976), Affinity Chromatography, Lowe and Dean, John Wiley and Sons (New York 1974), and Clin. Chem. 22:726 (1976); and for specific references see Science 144:1344 (1967) [the carbodiimide reaction], Erlanger et al, Methods in Immunology and Immunochemistry, ed. Williams and Chase, Academic Press (New York 1967), p. 149 [the mixed anhydride reaction], Peptides and Amino Acids, Kopple, W. A. Benjamin, Inc. (New York 1966) [the acid azide and active ester reactions], and Proc. Nat. Acad. Sci. USA 66:651 (1970) [the bis-imidate reaction].